Genetic Structure and Population Differentiation of the Mediterranean Pioneer Spiny Broom Calicotome Villosa Across the Strait of Gibraltar

Genetic structure and population differentiation of the Mediterranean pioneer spiny broom Calicotome villosa across the Strait of Gibraltar

JUAN ARROYO*, ABELARDO APARICIO, RAFAEL G. ALBALADEJO, JOAQUÍN MUÑOZ† and RITA BRAZA

Departamento de Biología Vegetal y Ecología, Universidad de Sevilla, Apartado 1095, E-41080 Sevilla, Spain

The region around the Strait of Gibraltar is considered to be one of the most relevant ‘hot spots’ of biodiversity in the Mediterranean Basin due to its historical, biogeographical, and ecological features. Prominent among these is its role as a land bridge for the migration and differentiation of species during the Pleistocene, as a consequence of the lowering of sea level and climate changes associated with the Ice Ages. In the present study, we report a multilevel hierarchical investigation of the genetic diversity of Calicotome villosa, a common pioneer legume shrub, at the regional scale. The results of genetic analysis of progeny arrays are consistent with a predominantly outcrossing mating system in all the populations analysed. Geographically, a pattern of population isolation by distance was found, but the Strait accounted for only approximately 2% of the among-population genetic differentiation. Consequently, extensive historical gene flow appears to be the rule for this species in this area. According to the natural history traits of C. villosa (pollination, dispersal, and colonization ability), we hypothesize that gene flow must be strongly influenced by seed dispersal because pollen flow is very limited. Based on the history of trade and land use, cattle and human movements across the Strait must have strongly favoured seed dispersal. We review and discuss these results and compare them with those of other reported studies of genetic and phylogenetic differentiation across the Strait of Gibraltar. It is stressed that colonization ability, which depends upon seed dispersal and life form, can be a more critical factor in gene flow than pollination. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 93, 39–51.

ADDITIONAL KEYWORDS: allozymes – breeding system – colonization – dispersal – gene flow – legume – Mediterranean biogeography – population genetics.

INTRODUCTION of land bridges attempted to explain global patterns of biogeographical similarities (for a botanical per- In biogeography, some areas are of particular interest spective, see van Steenis, 1962) until it was super- because their geographical settings and historical seded by interpretations based on continental drift constraints are thought to strongly influence the (Raven & Axelrod, 1974). Some particular areas, such geographical ranges of species. Among these areas, as Beringia and Mesoamerica, are still considered as islands, refugia, and corridors are favourite targets appropriate places to study the effects of land bridges because of the strong limitations on species move- as corridors for historical species movements. This is ments, making it easier to interpret observed pat- hypothesized to be the most plausible explanation of terns (Hewitt, 2000; Holloway, 2003). The old theory the current range of several animal and plant species

(Cavers, Navarro & Lowe, 2003; Brubaker et al., 2005; García-Moreno et al., 2006). One of these areas *Corresponding author. E-mail: [email protected] †Current address: Estación Biológica de Doñana, CSIC, is split by the Strait of Gibraltar, which separates the Sevilla, Spain. Eurasian and African plates at their westernmost

extremities. This region has had a convulsive geologi- been suggested in two other pioneer shrub legumes, cal history, involving recurrent closings and openings Cytisus and Retama (Parker, 1997; Rodríguez-Riaño, of the Strait until the late Tertiary, 5.5 Mya (Duggen Ortega-Olivencia & Devesa, 1999a), the closest rela- et al., 2003). Although there was no physical connec- tives of Calicotome (Käss & Wink, 1997; Cubas, Pardo tion during the Pleistocene, the Ice Ages provoked a & Tahiri, 2002). lowering of the sea level, resulting in the land masses In the present study, we performed a multilevel being closer. It has been stressed that this complex hierarchical investigation of the genetic diversity, history, in terms of its geological, climatic, and tem- genetic identity, and mating system in populations of poral heterogeneity, has determined most of the evo- C. villosa at the regional scale of the Strait of Gibral- lution of the plant lineages in the Mediterranean tar. The aim was to compare the observed patterns (Thompson, 2005). This geological instability, together with those previously reported for other species with with Pleistocene climate changes, may have increased different ecological requirements, contrasting biologi- the role of the Strait of Gibraltar as an active land cal traits (growth form, pollination and dispersal bridge in the migration and differentiation of species mechanisms) and historical and geographical ranges (Quézel, 1978; Caujapé-Castells & Jansen, 2003). across the Strait of Gibraltar. Accordingly, we should A comprehensive biogeographical study of the be able to test the extent to which the Strait has regional flora is lacking, but studies focusing on the represented a barrier to gene flow and promoted standing components of biodiversity and the ecology genetic differentiation. Our expectation was that, in a of woody communities across the Strait are available species with poor pollen dispersal, patterns of genetic (Ojeda, Marañón & Arroyo, 2000; Ajbilou, Marañón & differentiation should be related to seed dispersal and Arroyo, 2006). For example, short-term ecological pro- establishment, both of which are directly related to cesses associated with strong human disturbance colonizing ability (DeWoody, Nason & Smith, 2004). regimes (especially from the last millennium to the Specifically, given their suitability in reflecting his- present day; Mikesell, 1960; Reille, 1977; Moore et al., torical patterns of genetic differentiation and breed- 1998) explain the diversity of the lesser shrub and ing systems (Hamrick & Godt, 1989), we used tree species observed in northern Morocco relative to allozyme variation to determine: (1) the genetic diver- that in southern Spain (Ojeda, Marañón & Arroyo, sity, genetic structure, and genetic identity of C. 1996a; Ajbilou et al., 2006). On an evolutionary time villosa at both the population and regional scales and scale, most phylogenetic and phylogeographical (2) the breeding system of the species in selected analyses undertaken of woody species (Arroyo et al., populations using a mixed-mating model approach 2004; Rodríguez-Sánchez et al., in press) have shown (Ritland, 2002) in relation to its migration ability. some genetic differentiation across the Strait of Finally, we discuss the results obtained in the light of Gibraltar, irrespective of taxonomic group, plant life the evidences gathered for other species in this same form, or reproductive traits. However, none of these region of high biogeographical significance. studies included widespread, pioneer species, whose large geographical ranges are due to their wide ecological niches (Morin & Chuine, 2006). Such a study MATERIAL AND METHODS would be useful to compare the effects of colonization ability on gene flow and on patterns of genetic varia- STUDY SPECIES tion at the regional scale. Calicotome Link [Cytiseae (= Genisteae), Legumino- Breeding systems and ecological requirements are sae] comprises four species of spiny brooms with reputedly associated with colonization and migration Mediterranean distributions. The monophyly of this processes (Baker & Stebbins, 1965), and species with genus and its genetic differentiation from other contrasted colonizing ability are particularly useful to genera have been demonstrated by Cubas et al. test these associations. Calicotome villosa (Poir.) Link (2002). Calicotome villosa is a circun Mediterranean is a common pioneer shrub, the geographical range component of the lowland shrub vegetation in open of which extends across both sides of the Strait of woodlands (Greuter, Burdet & Long, 1984–89). Gibraltar. This species usually grows in large, dense, Within the western Mediterranean area, the species and continuous populations in anthropogenic environ- is much more frequent in southern Spain and north- ments of intense land use (agriculture, herding). western Morocco, where it sometimes forms almost Although many ecological data are available for this monospecific shrublands. In this range, C. villosa is species (Ojeda, 1995; Ajbilou, 2001), detailed informa- associated with disturbance processes, mostly caused tion about its demography and reproductive biology by cattle browsing, and to a lesser extent by fire and remains very scarce and the breeding system of slashing. This species is very digestible by cattle C. villosa in particular remains unexplored. Partial (Anmar, López & González, 2005) and thus it occurs self-incompatibility or inbreeding depression has in open sites with low or no tree cover and extensive

Figure 1. Distribution areas of Calicotome villosa on both sides of the Strait of Gibraltar (shaded areas) and the locations of the 26 populations studied.

cattle management (cows and goats). However, the Mejías, Arroyo & Marañón, 2007) typically restricted species is quite resilient to serious damage because of to deep, wet, and temperate gorges. On the other strong spiny branches and profuse regeneration by hand, open lowlands of fertile soils harbour most of seeds (R. Braza & J. Arroyo, unpubl. data). The the typical, widespread Mediterranean species species’ tolerance is promoted by strong resprouting (Ajbilou, 2001). after damage. Calicotome villosa has a pollination mechanism usually triggered by bees of medium to large size (Arroyo, 1981; Rodríguez-Riaño, Ortega- FIELD SAMPLING AND ALLOZYME ELECTROPHORESIS Olivencia & Devesa, 1999b). The species is potentially Twenty-six populations of C. villosa were sampled dispersed by cattle when browsed during the fruiting across the Strait of Gibraltar, including nine in season. Humans may also facilitate dispersal because Morocco and 17 in Spain (Table 1, Fig. 1). The densi- the species is more frequent along roadsides and ties of the populations sampled in the study area paths. Furthermore, the species survives the dry approximately reflect the natural abundance of popu- summer period because of its summer-deciduous lations, with more samples taken in areas close to the habit (DeLillis & Fontanella, 1992). Strait, and less in distant areas. This distribution is in part due to the fact that natural vegetation is steadily replaced by cultivated lands in low-altitude STUDY AREA areas where the studied species grows. The region of the Strait of Gibraltar is considered The vegetative tissues of C. villosa (leaves and coty- one of the most important ‘hot spots’ of biodiversity ledons) were discarded for electrophoresis because of in the Mediterranean Basin (Médail & Quézel, their poor resolution and inconsistency in zymograms. 1997). On the one hand, geological instability and Instead, seeds were collected and the embryos used climatic changes (Duggen et al., 2003; Pirazzoli, for electrophoresis. In 1993 and 1994, seeds were 2005) have actively shaped the floristic and commu- picked from 30 mother plants in each population nity compositions, giving rise to a rich endemic whenever possible and stored at 4 °C in dark until woody flora restricted to poor sandstone-derived their proteins were extracted. Electrophoreses were soils (e.g. Satureja salzmannii, Teline tribracteolata, run using one seed per mother plant, with 30 seeds Drosophyllum lusitanicum, Thymelaea villosa, per population (i.e. 30 mothers per population). In the Genista tridens; Arroyo, 1997; Médail & Quézel, small Moroccan populations M90 and M99, two to 1997; Ojeda et al., 2000) and a group of late Tertiary three seeds were used from each available mother and Pleistocene relict species (e.g. Rhododendron plant (16 and 11, respectively) to complete 30 seeds ponticum, Frangula alnus; Hampe et al., 2003; per population.

Table 1. Allele frequencies for polymorphic loci of Calicotome villosa at the population and regional levels (M = Morocco, E = Spain), and for all populations combined

Populations

Locus Allele M5 M6 M10 M87 M89 M90 M91 M99 M100 E68 E69 E70 E71 E72

Adh-1 A 0.800 0.883 0.950 0.917 0.917 0.983 0.967 0.700 1.000 0.967 0.933 0.967 0.967 0.917 B 0.200 0.117 0.050 0.083 0.083 0.017 0.033 0.300 – 0.033 0.067 0.033 0.033 0.083 6-Pgd-1 A 0.317 0.233 0.100 0.104 0.500 – 0.083 0.182 0.179 0.294 0.207 0.261 0.217 0.240 B 0.683 0.767 0.900 0.896 0.500 1.000 0.917 0.818 0.821 0.706 0.793 0.739 0.783 0.760 6-Pgd-2 A 0.983 0.950 1.000 0.519 0.904 1.000 0.500 0.955 1.000 0.966 1.000 0.981 0.917 0.920 B 0.017 0.050 – 0.481 0.096 – 0.500 0.046 – 0.034 – 0.019 0.083 0.080 Pgm-1 A 0.345 0.357 0.313 0.283 0.407 0.350 0.500 0.433 0.333 0.450 0.417 0.052 0.450 0.367 B 0.655 0.643 0.688 0.717 0.593 0.650 0.500 0.567 0.667 0.550 0.583 0.948 0.550 0.633 Pgm-2 A 0.143 0.183 0.080 0.167 0.172 0.200 0.067 0.286 0.183 0.450 0.300 0.362 0.067 0.167 B 0.786 0.683 0.920 0.783 0.793 0.783 0.850 0.714 0.783 0.533 0.700 0.569 0.467 0.717 C 0.071 0.133 – 0.050 0.035 0.017 0.083 – 0.033 0.016 – 0.069 0.467 0.117

The seeds were scarified and soaked in distilled Spain) levels using the Genetic Data Analysis program water overnight. The seed coat and the endosperm (GDA; Lewis & Zaykin, 2001), including the mean layer were then removed and discarded, and the number of alleles per locus (A), percentage of polymor- embryos homogenized in four drops of DL-dithiothreitol phic loci (P, at the 99% cut-off), mean number of alleles

(0.065 M) and Na2HPO4 (0.05 M) buffer at pH 7. Crude per polymorphic locus (Ap), and observed (Ho) and extracts were adsorbed onto Whatman 3MM paper expected (He) heterozygosity under Hardy–Weinberg wicks and stored at -80 °C until electrophoresis. Elec- equilibrium. Weir & Cockerham’s (1984) inbreeding trophoresis was performed following the general pro- coefficient (f) was computed for each population and its tocols of Wendel & Weeden (1989) on 12% starch and statistical significance was examined with a c2 test (Li 2.5% sucrose gels. Sixteen enzyme systems were tested & Horviz, 1953): c2 = f 2N(k – 1), with d.f. = k(k – 1)/2, for activity and consistent banding patterns in three where N is the number of individual plants sampled different electrode buffers following the staining and k is the number of alleles in the population. recipes of Soltis et al. (1983). Only three gave sufficient Population differentiation (q) was also computed consistent resolution in a histidine/citrate electrode using GDA. The statistics f and q are similar to buffer (pH = 6.5/6.5) (Cardy et al., 1983): alcohol Wright’s (1951) F-statistics and are unbiased with dehydrogenase (ADH; EC 1.1.1.1), phosphogluconate respect to small and uneven sample numbers and dehydrogenase (6-PGD; EC 1.1.1.43), and phosphoglu- population sizes (Culley & Grubb, 2003). The inbreed- comutase (PGM; EC 5.4.2.2). Zymograms were inter- ing coefficient is a measure of the correlation of the preted in terms of loci and alleles, which were labelled genes of individuals within populations and is conse- starting from the most anodally migrating band. quently analogous to Fis, whereas q is a measure of the amount of differentiation among populations rela- MATING-SYSTEM ESTIMATES tive to the total diversity and is therefore analogous to Fst. q was calculated across loci at the regional level Sampling included the simultaneous collection of seed in Morocco and Spain (qpM and qpS, respectively), families for mating-system estimations. In particular, among populations within regions (qp), and between populations E68, M89, E81, and E84 (for locations, regions (qr). To evaluate the relative influences of see Fig. 1) were selected for this purpose because they genetic drift and historical gene flow on the distribu- provided both adequate numbers of seeds and high tion of genetic variation, assuming a stepping-stone or low Ho values. Ten pooled seeds from each of 15 model of population structure (Hutchinson & Temple- mothers randomly selected in each population were ton, 1999), q was also calculated for each possible pair screened for allozyme variation (i.e. 10 seeds ¥ of populations using GDA. Their correlation with geo- 15 families ¥ 4 populations). All five polymorphic loci graphical distances was evaluated with the Mantel that were resolved (see Results) were used for this test in GENETIX, version 4.02 (Belkhir et al., 2001). purpose. Slightly negative values of q were set to zero (Williams & Guries, 1994). A population pairwise DATA ANALYSIS geographical distance matrix was generated using Genetic diversity statistics were hierarchically calcu- ArcGis procedures (Environmental Research Insti- lated at both the population and regional (Morocco and tute, Redlands, CA, USA).

Populations All E73 E74 E75 E76 E77 E78 E79 E81 E82 E83 E84 E85 Morocco Spain populations

1.000 0.917 0.817 0.950 0.967 0.950 0.967 0.967 0.967 0.983 1.000 1.000 0.902 0.955 0.937 – 0.083 0.183 0.050 0.033 0.050 0.033 0.033 0.033 0.017 – – 0.098 0.045 0.064 0.259 0.317 0.433 0.339 0.214 0.103 0.150 0.217 0.375 0.183 0.133 0.367 0.191 0.254 0.233 0.741 0.683 0.567 0.661 0.786 0.897 0.850 0.783 0.625 0.817 0.867 0.633 0.809 0.746 0.767 0.948 0.933 0.850 0.946 0.982 0.862 0.917 1.000 1.000 0.967 0.967 0.967 0.870 0.947 0.922 0.052 0.067 0.150 0.054 0.018 0.138 0.083 – – 0.033 0.033 0.033 0.130 0.053 0.078 0.450 0.350 0.109 0.160 0.400 0.417 0.217 0.067 0.259 0.217 0.207 0.383 0.370 0.297 0.322 0.550 0.650 0.891 0.840 0.600 0.583 0.783 0.933 0.741 0.783 0.793 0.617 0.630 0.703 0.678 0.150 0.417 0.200 0.111 0.233 0.167 0.233 0.067 0.077 0.150 0.133 0.017 0.165 0.195 0.185 0.750 0.567 0.733 0.870 0.433 0.567 0.550 0.433 0.846 0.783 0.817 0.333 0.786 0.625 0.680 0.100 0.017 0.067 0.019 0.333 0.267 0.217 0.500 0.077 0.067 0.050 0.650 0.048 0.180 0.135

Nei’s (1972) genetic identity and distance were also 6-Pgd-1, 6-Pgd-2, and Adh-1 were fixed in at least one calculated with GDA, and the linkage disequilibrium Moroccan population whereas 6-Pgd-2 and Adh-1 of individual loci and the effective number of alleles were fixed in at least one Spanish population. Allele per locus (Ne) were estimated using POPGENE, frequencies were not significantly different between version 1.32 (Yeh & Boyle, 1997). A Neighbour-joining Morocco and Spain, and no alleles were exclusive at (NJ) tree of populations based on Nei’s (1972) genetic either the regional or population level (Table 1). No distances was constructed and branch support linkage disequilibrium was detected between loci assessed by bootstrapping (1000 replicates) with within any of the populations. PHYLIP, version 3.5 (Felsenstein, 1993). Measures of genetic variation did not differ sig- For progeny, maximum likelihood multilocus and nificantly at the population or regional level. The single-locus outcrossing rates (tm, ts), correlated pater- percentage of polymorphic loci was in the range nity (rp), and biparental inbreeding (tm – ts) were com- 50–83.3% but the number of alleles per locus was very puted following Ritland’s (1989) mixed-mating model low (range 1.66–2.00), and the number of alleles per and sibling-pair model with MLTR, version 3.0 polymorphic locus was also low (ranging 2.20–2.33). (Ritland, 2002). Standard errors were calculated by Most loci consisted of one frequent allele, so the bootstrapping over families (1000 replicates). Because effective number of alleles was in the range 1.208– most seeds could be assigned to a specific genotype, 1.561 (Table 2). we applied the Newton–Raphson algorithm, which At the population level, most populations (81%) provides estimates of tm from the theoretical bounds exhibited an excess of heterozygotes. This excess sig- when dealing with multiple heterozygote fathered nificantly differed from Hardy–Weinberg expectations progeny arrays (Ritland, 2002). in two populations in Morocco (M89 and M91) and in For each population, the maternal multilocus geno- two in Spain (E68 and E71) (Table 2). Across loci, types inferred by MLTR were used to compute the Pgm-1 showed a significant excess of heterozygotes, ‘observed’ maternal inbreeding coefficient (fob) using whereas Pgm-2 showed a significant deficiency. The GDA. ‘Expected’ maternal inbreeding (fex) was calcu- same trend for these two loci was observed in both lated with the equation tm = (1 - fex)/(1 + fex) (Jain, the Spanish and Moroccan subsets of populations. 1979), given the tm population outcrossing rate. 6-Pgd-2 showed a significant excess of heterozygotes

in Morocco (Table 3). Finally, the overall inbreeding RESULTS coefficient (f) was not significantly different from zero at either the regional or species level, showing no GENETIC DIVERSITY AND POPULATION departure from Hardy–Weinberg proportions. GENETIC STRUCTURE Between-population genetic identities were high, ADH, 6-PGD, and PGM, encoded by six loci (two loci ranging between 0.999 (M6/E72) and 0.866 (M91/E85) each), showed consistent and interpretable bands on (overall mean ± SD = 0.965 ± 0.025). At the regional all zymograms. Adh-2 was monomorphic across all scale, populations in Morocco and in Spain showed populations and the rest of the loci were polymorphic exactly the same mean values. Accordingly, the NJ in at least 20 populations. At the regional scale, tree based on Nei’s (1972) genetic distances showed

Table 2. Genetic diversity statistics for populations, regions and all populations combined of Calicotome villosa ARROYO

Populations N A P Ap Ne Ho He f

ET M5 29.5 2.000 83.3 2.200 1.441 0.306 0.270 -0.134

M6 29.7 2.000 83.3 2.200 1.450 0.274 0.271 -0.010 AL. © M10 28.2 1.667 66.7 2.000 1.208 0.167 0.145 -0.157

2008 2008 M87 28.3 2.000 83.3 2.200 1.441 0.321 0.272 -0.185 M89 28.0 2.000 83.3 2.200 1.473 0.397 0.280 -0.428*

The The M90 27.7 1.667 50.0 2.333 1.233 0.117 0.141 0.177

Linnean M91 29.3 2.000 83.3 2.200 1.435 0.348 0.252 -0.394* M99 27.0 1.833 83.3 2.000 1.483 0.332 0.289 -0.152

M100 29.3 1.667 50.0 2.333 1.293 0.193 0.185 -0.045

Society E68 29.7 2.000 83.3 2.200 1.480 0.365 0.263 -0.395* E69 29.8 1.667 66.7 2.000 1.384 0.308 0.230 -0.344 E70 27.8 2.000 83.3 2.200 1.337 0.196 0.191 -0.027 of E71 30.0 2.000 83.3 2.200 1.503 0.400 0.273 -0.476**

London, E72 28.3 2.000 83.3 2.200 1.433 0.273 0.267 -0.024 E73 29.7 1.833 66.7 2.250 1.399 0.281 0.234 -0.205 E74 30.0 2.000 83.3 2.200 1.490 0.289 0.283 -0.021

Biological E75 28.8 2.000 83.3 2.200 1.449 0.303 0.281 -0.079 E76 28.0 2.000 83.3 2.200 1.283 0.194 0.194 0.002 E77 29.3 2.000 83.3 2.200 1.561 0.327 0.265 -0.241

E78 29.7 2.000 83.3 2.200 1.495 0.314 0.269 -0.172 Journal Journal E79 30.0 2.000 83.3 2.200 1.430 0.244 0.239 -0.025 E81 27.7 1.833 66.7 2.250 1.331 0.161 0.185 0.128

E82 28.2 1.833 66.7 2.250 1.325 0.249 0.202 -0.239 of

E83 30.0 2.000 83.3 2.200 1.268 0.178 0.186 0.043 the the E84 29.8 1.833 66.7 2.250 1.219 0.119 0.159 0.256

Linnean E85 30.0 1.833 66.7 2.250 1.451 0.300 0.249 -0.210 Morocco 28.6 ± 0.94 1.871 ± 0.16 74.1 ± 15 2.185 ± 0.12 1.384 ± 0.11 0.273 ± 0.09 0.234 ± 0.06 -0.170 ± 0.19 Spain 29.2 ± 0.88 1.931 ± 0.10 77.5 ± 8 2.203 ± 0.06 1.402 ± 0.10 0.265 ± 0.08 0.234 ± 0.04 -0.140 ± 0.19

Society All populations 29.0 ± 0.94 1.910 ± 0.13 76.3 ± 11 2.197 ± 0.08 1.396 ± 0.10 0.268 ± 0.08 0.234 ± 0.05 -0.150 ± 0.19

, F-values significantly different from zero at *P < 0.05, **P < 0.001.

2008, Measures of genetic variation include sample size averaged over loci (N), mean number of alleles per locus (A), percentage of polymorphic loci (P, 99% cut-off),

mean number of alleles per polymorphic locus (Ap), effective number of alleles per locus (Ne), observed heterozygosity (Ho), expected heterozygosity (He), and the

93 fixation index (f) of Weir & Cockerham (1984). ,

39 – 51

Table 3. F-statistics for both regions (Morocco and Spain) and for all populations combined of Calicotome villosa, including the fixation index of Weir & Cockerham (1984) (f), the proportion of genetic diversity partitioned among populations within regions (qpM and qpS for Morocco and Spain, respectively), and the proportion of genetic diversity partitioned among populations (qp) and regions (qr) in the species (all populations)

Morocco Spain All populations

Locus f qpM f qpS f qp qr

Adh-1 -0.2074 0.0923 -0.0758 0.0291 -0.1425 0.0768 0.0172 6-Pgd-1 -0.3946 0.1269 -0.3493 0.0340 -0.3617 0.0649 0.0047 6-Pgd-2 -0.6494 0.3521 -0.0790 0.0240 -0.3293 0.2148 0.0236 Pgm-1 -0.4126 0.0091 -0.4822 0.0844 -0.4554 0.0636 0.0063 Pgm-2 0.5628 0.0027 0.2762 0.1301 0.3559 0.1313 0.0350 Mean -0.1667 0.0934 -0.1400 0.0811 -0.1487 0.1007 0.0175 95% CIlower -0.4819 0.0147 -0.4173 0.0302 -0.4129 0.0650 0.0064 95% CIupper 0.3029 0.2279 0.2193 0.1170 0.2323 0.1502 0.0317

Upper and lower confidence intervals (CI) for mean values were estimated by bootstrapping (2000 replicates) across loci.

no pattern of geographical clustering of populations were consistently low and correlated paternity (rp) across the Strait of Gibraltar and very low bootstrap was markedly different among populations, with support for most branches (Fig. 2). 8–71% of seeds within a family being full sibs.

Within each region, the genetic differentiation The observed maternal inbreeding coefficient (fob) among populations was not great. Overall, the was in the range -0.276 to 0.176, whereas the population genetic differentiation was moderate expected maternal inbreeding coefficient (fex) was in

(qp = 0.101). Therefore, approximately 90% of the the range 0.00–0.234. When we compared fob with fex, genetic variation occurred within populations. At the a deficit of homozygotes became evident in the four regional scale, the Strait of Gibraltar can only populations studied, which was remarkably high in account for 1.8% of the total genetic variation of populations E81 and M89 (Fig. 4).

C. villosa (qr = 0.018) (Table 3). The genetic differentiation in all the studied populations was significantly correlated with geographical distance (r = 0.444, DISCUSSION P = 0.004), although the correlation coefficient decreased, but was still significant, when the geo- In the present study, we have shown that the Strait of graphically most distant population (E85) was Gibraltar has not acted as a biogeographical barrier excluded from the analysis (r = 0.241, P = 0.027). At for C. villosa on the historical time scale, as demon- the regional scale, the Moroccan populations did strated by the lack of genetic structure across the not show a significant correlation between pairwise studied populations on both sides of the Strait. Some q-values and geographical distance (r = -0.230, ecological and biological traits of C. villosa may P = 0.862), whereas all Spanish populations showed a explain the observed results. significant correlation (r = 0.535, P = 0.010), robust to the inclusion of the distant E85 population (r = 0.224, P = 0.048). Under the assumption of a stepping-stone POPULATION GENETIC STRUCTURE, MATING SYSTEM, population model, in which gene flow is most likely to AND DISPERSAL occur between neighbouring populations, the scatter Most scored loci were polymorphic, with the number plot of q versus geographical distance is consistent of alleles per polymorphic locus (Ap) and the effective with a lack of equilibrium between gene flow and number of alleles (Ne) per locus at the population genetic drift in C. villosa, with historical gene flow level being similar to the average values for many being much more influential than genetic drift other plant species with different life-history traits (Fig. 3). (Hamrick & Godt, 1989). The values for gene diversity

Multilocus outcrossing rates (tm) were high for (He) were high, consistent with averaged values for populations E68, E81, and M89 and moderate for long-lived perennial plants with effective seed- population E84 (Table 4), indicating a general trend dispersal mechanisms and outcrossing breeding towards outcrossing in the studied populations of systems (Hamrick & Godt, 1996). This is relevant C. villosa. The levels of biparental inbreeding (tm – ts) because reproductive biology, together with other life-

E81

E85

E71

E74 E77 59 E70

E68

59 E78 69 M5 E72

E79 M99 E69

E75

E73 M6

M100 M89 66 E83 E82 E76

E84

M87 M90

M10

0.01 changes M91

Figure 2. Unrooted Neighbour-joining tree for the 26 populations of Calicotome villosa studied across the Strait of Gibraltar based on Nei’s (1972) genetic distances. Branch support was calculated by bootstrap analysis (1000 replicates). Only values above 50% are shown.

history traits of species, is a keystone in interpreting large discrepancies and, consequently, no significant population genetic variation (Hamrick & Godt, 1989). departure from Hardy–Weinberg expectations was Self-incompatibility is relatively common in the detected at the species level. Despite the overall nega- Leguminosae, especially among woody species (Arroyo, tive value of the inbreeding coefficient (f) at the popu- 1981). Within the tribe Cytiseae, low fruit set after lation level, only four populations (15.4%) showed a self-pollination has been interpreted in some species of significant departure from random mating, with an Cytisus and Retama as partial self-incompatibility excess of heterozygotes. or late-acting inbreeding depression (Parker, 1997; In C. villosa, we found high values of He and low Rodríguez-Riaño et al., 1999a). Detailed studies of the values of q, typical of a species with potential long- reproductive biology of C. villosa are lacking but the distance dispersal events in seed flow and/or pollen genetic data collected in the present study on seed flow (Hamrick & Godt, 1989, 1996). These events are progenies at the population level reveal high rates of dependent on the pollination and breeding systems, outcrossing (tm), supporting a major role for partial seed dispersal, habitat requirements, and the physi- self-incompatibility in the breeding system of this cal setting (barriers, habitat availability). First, species. In the present study, it was found that honey bees have often been observed visiting many observed and expected heterozygosities did not exhibit flowers in the mass flowering plants of C. villosa

1.0

Mantel test r=0.444, P=0.004

0.8 )

on( θ

i t a

i 0.6

ent

ffer i

d 0.4

i t

en G 0.2

0.0 0 40 80 120 160 200 240 280 Geographical distance (km)

Figure 3. Mantel test, correlation coefficient and associated P-value, and scatterplot showing the relationship between pairwise genetic differentiation among populations of Calicotome villosa (qp) in the region of the Strait of Gibraltar, and the geographical distances among them.

Table 4. Multilocus outcrossing rates (tm) and levels of biparental inbreeding (tm – ts) in the four populations of Calicotome villosa selected for progeny analysis (for details, see Material and methods)

Population tm tm - ts rp fob (95% CI)

E68 1.200 (0.028) 0.052 (0.043) 0.153 (0.274) -0.098 (-0.556–0.292) E81 0.824 (0.129) 0.097 (0.054) 0.707 (0.333) -0.236 (-0.471–0.104) E84 0.620 (0.142) -0.017 (0.034) 0.389 (0.362) 0.176 (-0.135–0.427) M89 0.875 (0.116) 0.050 (0.061) 0.086 (0.244) -0.267 (-0.450–0.180)

Standard errors based on bootstrap analysis (1000 replicates) are shown in parenthesis. CI, confidence interval.

plants (J. Arroyo, pers. observ.), as has also been 0.3 observed in related plant species in the region with 0.2 similar flower morphologies (Herrera, 1988). Thus, 0.1 among-population gene flow by pollen movement should be low. Second, explosive pods, which have a

f 0 limited dispersal distance, would also produce this

-0.1 pattern of mating. Many species have dispersal

mechanisms that are not directly related to their -0.2 inferred functional morphology (i.e. dispersal syn- -0.3 dromes; Herrera, 2002). For example, capsules from E68 E81 E84 M89 some species of shrubby Cistus can be ingested and dispersed by deer that browse in similar communities Figure 4. Observed (fob, filled diamonds) and expected (Malo & Suárez, 1998). The germinability of the (fex, open diamonds) maternal inbreeding coefficients in the woody legume Cytisus scoparius also increases after four populations of Calicotome villosa selected for progeny cattle gut passage (Manzano, Malo & Peco, 2005). The analysis (for details, see Material and methods). strong association of C. villosa with extensive herding and its palatability to cattle (Anmar et al., 2005) before moving to nearby plants, causing predomi- could account for such seed dispersal over longer nantly geitonogamy and cross pollination with rela- distances, as has been observed in other woody tives, whereas the much less frequent bee species of legumes (Prosopis flexuosa, Campos & Ojeda, 1997; the genus Anthophora tend to move to more distant Acacia spp., Argaw, Teketay & Olsson, 1999). More-

over, it has been demonstrated that Macaronesian Strait appears to be crossed by genes and, although broom species, also apparently limited in their poten- the pattern could be consistent with pollen flow, this tial seed dispersal, have repeatedly colonized oceanic flow is most probably by seeds. A simultaneous study islands (Percy & Cronk, 2002). Third, the association with appropriate molecular, nuclear, and organellar of this broom species with disturbed, often cultivated markers is required to resolve this question. Given land and roadsides, may cause seed mixing with the short distances over which pollen is dispersed by agricultural crops, and thus subject it to movement bee pollinators and over which seeds are dispersed by along trade routes. This may account for the lack of autochory, and in secondary dispersal by cattle, it is genetic differentiation across short distances and probable that the genetic exchange between the explain why most genetic diversity in C. villosa is African and Iberian sides is recent and was aided by within rather than among populations, even when the humans (with the movement of cattle and crops). historical existence of the Strait of Gibraltar is taken There are very few studies of the patterns of genetic into account. Only at the longest distances within the differentiation of wild species mediated by human range does there appear to exist considerable genetic influence in the studied region, except when they are differentiation (as indicated by the Mantel test), prob- deliberately cultivated (Tuomi & Lumaret, 1998). In ably because the process of range expansion is dif- this context, seed dispersal is a necessary, but not fusive, which is consistent with a short-distance sufficient, condition to preclude genetic differentiation stepping-stone model. because seeds must germinate and seedlings become established. In this species, the seed germination rate is very high and seed viability long lasting, and seed- ROLE OF HISTORICAL FACTORS ling growth is very fast under greenhouse conditions The area around the Strait of Gibraltar is one of the (Braza, 2005). Although its behaviour under field con- centres of diversity of the tribe Cytiseae (Gómez- ditions is not well known, we hypothesize that the González et al., 2004). Many of the species of this colonization ability of the species is very high, as has tribe are endemic to the area, and many others, such been observed in other gorse and broom species in the as C. villosa, present their largest populations there area (Ojeda, Marañón & Arroyo, 1996b). because broom scrubland is one of the prominent This pattern of differentiation across the Strait of woody communities in the region (Ojeda, Arroyo & Gibraltar contrasts with that observed in other Marañón, 1995). Although there has been no compre- species. Although the available information for plants hensive phylogenetic or phylogeographical study of straddling the Strait of Gibraltar is still scarce, there Calicotome, a wider study that included related are interesting contrasting patterns. Traditionally, genera (Cubas et al., 2002) showed the monophyly the Iberian Peninsula has been considered as one of and clear genetic differentiation of Calicotome with the three main Mediterranean refugia (together with both nuclear and plastid markers, although the two the Italian and Balkan Peninsulas) for plants and samples of C. villosa differed in their nuclear animals in harsh climatic periods since the late Ter- sequences. It is interesting to note that, although tiary (Hewitt, 2000). This refugium should probably many species of Cytiseae are narrowly endemic in the be extended to include northern Morocco because its area, only a few are confined to one side of the Strait ecological and historical conditions are similar to (e.g. Teline tribracteloata on the Iberian side, Teline those in southern Spain (Reille, 1977). The Strait has osmariensis, on the Moroccan side). This may be due probably been a filter for some species through its to the ancient origin of these taxa before the separa- expansion/retraction cycles during the Quaternary, tion of the African and Iberian plates and an improb- insofar as the nearby African side has a tree and able lack of differentiation during the subsequent shrub flora poorer than that on the Spanish side 5.5 Mya, or to their dispersal across the Strait once it (Marañón et al., 1999; Ajbilou et al., 2006). This had formed. A recent study of closely related Ulex filtering process must involve the biological traits species on both sides of the Strait of Gibraltar, using and performance of species. Thus, some emblematic polymerase chain reaction-based plastid microsatel- long-lived species of Mediterranean forests (Quercus lite markers (Cubas, Pardo & Tahiri, 2005), which rotundifolia/ilex, Quercus suber, Pinus pinaster) and indicate gene flow by seed, showed that the differ- some relict species (Frangula alnus) show a clear ences among species are very low compared to the genetic discontinuity across the Strait, mostly attrib- differences among populations (on the long-distance utable to limited seed dispersal (Tuomi & Lumaret, dispersal of other broom species, see also Percy & 1998; Burban et al., 1999; Salvador et al., 2000; Cronk, 2002). Ulex spp. probably have seed dispersal Lumaret et al., 2002, 2005; Burban & Petit, 2003; patterns similar to those of Calicotome, which sug- Hampe et al., 2003). Although these species have gests that the Strait is not a biogeographical barrier fruits that are potentially dispersed by animals to these legume species. The 14-km width of the (Gómez, 2003; Hampe, 2004) or wind over longer

distances than are the seeds of C. villosa, they could an Acacia woodland of the Rift Valley in Ethiopia. Journal not cross this effective barrier. Unlike our study of Arid Environments 43: 411–435. species, those species are not associated with distur- Arroyo MTK. 1981. Breeding systems and pollination biology bances and their dispersal is not affected by human in leguminosae. In: Polhill RM, Raven PH, eds. Advances in activities, except when they are deliberately planted. legume systematics, Part 2. Kew: Royal Botanic Gardens, All of them are long-lived trees, late-successional 723–769. species with reduced colonizing ability compared to Arroyo J. 1997. Plant diversity in the region of the Strait of that of shorted-lived, early successional species. Gibraltar: a multilevel approach. Lagascalia 19: 393–404. Arroyo J, Carrión JS, Hampe A, Jordano P. 2004. La However, herbaceous species show a variety of pat- distribución de las especies a diferentes escalas espacio- terns, with no differentiation in the bulb geophyte temporales. In: Valladares F, ed. Ecología del bosque medi- Androcymbium gramineum (Caujapé-Castells & terráneo en un mundo cambiante. Madrid, España: Jansen, 2003), and strong differentiation in the Ministerio de Medio Ambiente, EGRAF, S.A., 27–67. perennial herb Saxifraga globulifera (Vargas, Morton Baker HG, Stebbins GL, eds. 1965. The genetics of coloniz- & Jury, 1999). Even within the same genus, Bellis ing species. New York, NY: Academic Press. annua (Compositae) shows genetic differentiation Belkhir K, Borsa P, Chikhi L, Raufaste N, Bonhomme F. among European and African populations, whereas 2001. GENETIX 4.02, logiciel sous Windows TM pour Bellis microcephala does not, although both species la génétique des populations. Montpellier: Laboratoire are annuals (Fiz, Valcárcel & Vargas, 2002). Because Génome, Populations, Interactions, CNRS UMR 5000, Uni- of the low number of species studied, we are far from versité de Montpellier II. determining the general properties of species filtered Braza R. 2005. ‘Fitness’ inicial en relación con las tasas de by the Strait. Nonetheless, it appears that the high cruzamiento y la heterocigosidad en Calicotome villosa seed dispersal and establishment potential of pioneer (Poir.) Link. (Leguminosae). MSc Thesis, University of shrub species are key factors suppressing their Seville. genetic differentiation. Brubaker LB, Anderson PM, Edwards ME, Lozhkin AV. 2005. Beringia as a glacial refugium for boreal trees and shrubs: new perspectives from mapped pollen data. Journal ACKNOWLEDGEMENTS of Biogeography 32: 833–848. Burban C, Petit RJ. 2003. Phylogeography of maritime pine We are grateful for the financial support of grants inferred with organelle markers having contrasted inherit- PB0551-95, PB1144-98BOS, 2003-07924-CO2-01 to ance. Molecular Ecology 12: 1487–1195. J.A., and the support of the Consejería de Medio Am- Burban C, Petit RJ, Carcreff E, Jactel H. 1999. Range biente (Andalusian Regional Government), CGL2004- wide variation of the maritime pine bast scale Matsucoccus 00022, and Fundación BBVA to A.A. Rosario Hidalgo feytandii Duc. (Homoptera: Matsucoccidae) in relation to the helped greatly with electrophoresis. Fernando Ojeda, genetic structure of its host. Molecular Ecology 8: 1593– Vicente Jurado, and Redouan Ajbilou shared with us 1602. their extensive data on the vegetation ecology of the Campos CM, Ojeda RA. 1997. Dispersal and germination of woodlands around the Strait of Gibraltar, and EVOCA Prosopis flexuosa (Fabaceae) seeds by desert mammals in group (http://www.alojamientos.us.es/grnm210) pro- Argentina. Journal of Arid Environments 35: 707–714. vided a stimulating atmosphere in which our results Cardy BJ, Stuber CW, Wendel JS, Goodman MM. 1983. were discussed. 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